Many established cell lines are available for a variety of purposes in biotechnology. Some cell lines can be cultivated as single-cell suspensions, but other cell lines do not grow well without a support. The growth of a cell line that requires support is often limited by the surface area available for the cells to grow on, since many cell lines will form only a monocellular layer on the surface. In addition, some cell lines may tend to grow in clumps or aggregates in the absence of a support, which is an undesirable result when they are needed as single-cell suspensions, but more especially when the cells are to be infected with a virus or transformed with a recombinant vector, since the virus or vector may not gain access to the cells within the clump or aggregate. Thus, there can be severe problems in scaling up the cultivation of a cell line, in particular in providing enough surface area for the cells to grow on and/or avoiding clumping of the cells.
Microcarrier technology has been used to cultivate cells in culture. For example, Forestell et al. (Biotech. Bioeng. 40: 1039-1044 (1992)) disclosed extended serial subculture of human diploid fibroblasts on microcarriers using a medium supplement that reduced the need of the cultured cells for serum. Furthermore, Ohlson et al. (Cytotechnology 14:67-80 (1994)) disclosed the bead to bead transfer of Chinese hamster ovary cells using macroporous gelatin microcarriers. Finally, Hu et al. (Biotech. Bioenc. 27: 1466-1476 (1985)) disclosed the serial propagation of mammalian cells on microcarriers using a selection pH trypsinization technique.
However, in view of the problems noted above, there is a need for improvements in methods of cultivating cell lines, in methods of producing viruses for clinical uses, and, in methods of scaling up the production of viruses for larger-scale commercialization. The instant invention meets these needs and more.
One aspect of the invention is a method for cultivating cells, comprising:
(a) cultivating the cells on a first batch of microcarriers until the cells are substantially confluent;
(b) detaching the cells from the microcarriers without removing the microcarriers from suspension;
(c) Adding a second batch of microcarriers; and
(d) cultivating the cells further.
Another aspect of the invention is a method of detaching cells from a first batch of microcarriers comprises the following steps:
(a) washing the microcarriers and attached cells to remove soluble materials;
(b) contacting the microcarriers and washed cells with a chelating agent;
(c) removing the chelating agent;
(d) trypsinizing the cells for a short period to detach the cells from the microcarriers; and
(e) neutralizing the trypsin by adding protein,
wherein (a)-(e) are conducted in a single cultivation vessel.
A further aspect of the invention is a method for the separation of cells from microcarriers on which they have been cultivated but from which they have become detached, comprising introducing an aqueous suspension of cells and microcarriers through an inlet into a separation device, the device comprising
(a) an inlet;
(b) a column;
(c) an outlet for the collection of cells and the aqueous solution; and
(d) a mesh screen;
wherein the microcarriers are retained in suspension by an upward flow in the separation device and are retained in the separation device by a mesh screen, and wherein the cells and aqueous solution are collected through the outlet.
A further aspect of the invention is a system for separating cells from microcarriers on which the cells have been cultivated, the system comprising;
(a) a bioreactor in which the cells were cultivated on the microcarriers;
(b) a flow path from the bioreactor to a separation device;
(c) a separation device comprising
(i) an inlet;
(ii) a column;
(iii) an outlet for the collection of cells and the aqueous solution; and
(iv) a mesh screen;
wherein the microcarriers are retained in suspension by the upward flow in the separation device and are retained in the separation device by a mesh screen, and the cells and aqueous solution are collected through the outlet; and
(d) a pump, wherein the pump directs the flow of the aqueous solution from the bioreactor to the outlet.